Targeted next generation sequencing assays for DNA level alterations such as point mutations and copy number alterations are routinely used for patients with advanced cancer considering clinical trials for investigational agents. RNA sequencing, while successfully applied for the discovery of gene fusions and quantitative assessment of gene expression, has yet to be translated into clinical grade testing for patient care in part due to limited expertise and perceived difficulty in creating an accurate and precise assay in a clinical grade laboratory. We hypothesize that targeted RNA sequencing can serve as a versatile clinical grade tool for molecular eligibility of patients with advanced cancer for therapeutic trials. In this proposal, we will develop a clinical grade targeted RNA sequencing assay including analytic validation (Aim 1: Accuracy, Precision Aim 2: Performance Range) and performance on clinical real world samples (Aim 3: Clinical Validation), and finally we will broadly distribute this assay by providing probes/protocols (Aim 4: Distribution of reagents and protocols). Targeted RNA sequencing is an unbiased strategy compared to traditional FISH or PCR assays, but because it is focused on actionable genes. It is also cost-effective for routine use in pathology labs on desktop sequencing instruments for any solid tumor or hematologic malignancy. During the UH2 Phase of Analytic Validation, we will determine the sensitivity and specificity of targeted RNAseq to detect gene fusions utilizing serial dilutions. This will include delineation of reportable ranges and intervals on control samples. Performance on various quantities of RNA, degraded RNA, frozen and formalin-fixed paraffin- embedded samples will be assessed. Reproducibility (between runs) and repeatability (within runs) will be assessed to define assay precision. During the UH3 Phase of Clinical Validation, the assay will be applied to a diverse collection of samples comprising known gene fusions including varied kinases and fusion structures. Further, UH3 will include performance assessment on unknown tumor samples collected from an active clinical tumor sequencing study. Lastly, the assay will be exported to another cancer center to expand clinical validation of the assay and to demonstrate how the assay can be exported for broader use in patient care. We are well suited to carry out this proposal through an established CLIA-certified Cancer Genomics Laboratory and a multi-disciplinary team including medical oncologists, bioinformaticians, pathologists, clinical laboratorians. This proposal will have a broader impact in clinical trials for oncology by demonstrating the clinical validity and utility of RNA sequencing for clinical decision-making.